Annexins A2 and A6 interact with the extreme N terminus of tau and thereby contribute to tau's axonal localization.

During neuronal development, the microtubule-associated protein tau becomes enriched in the axon, where it remains concentrated in the healthy brain. In tauopathies such as Alzheimer's disease, tau redistributes from the axon to the somatodendritic compartment. However, the cellular mechanism that regulates tau's localization remains unclear. We report here that tau interacts with the Ca2+-regulated plasma membrane-binding protein annexin A2 (AnxA2) via tau's extreme N terminus encoded by the first exon (E1). Bioinformatics analysis identified two conserved eight-amino-acids-long motifs within E1 in mammals. Using a heterologous yeast system, we found that disease-related mutations and pseudophosphorylation of Tyr-18, located within E1 but outside of the two conserved regions, do not influence tau's interaction with AnxA2. We further observed that tau interacts with the core domain of AnxA2 in a Ca2+-induced open conformation and interacts also with AnxA6. Moreover, lack of E1 moderately increased tau's association rate to microtubules, consistent with the supposition that the presence of the tau-annexin interaction reduces the availability of tau to interact with microtubules. Of note, intracellular competition through overexpression of E1-containing constructs reduced tau's axonal enrichment in primary neurons. Our results suggest that the E1-mediated tau-annexin interaction contributes to the enrichment of tau in the axon and is involved in its redistribution in pathological conditions.

An evolutionary roadmap to the microtubule-associated protein MAP Tau.

BACKGROUND: The microtubule associated protein Tau (MAPT) promotes assembly and interaction of microtubules with the cytoskeleton, impinging on axonal transport and synaptic plasticity. Its neuronal expression and intrinsic disorder implicate it in some 30 tauopathies such as Alzheimer's disease and frontotemporal dementia. These pathophysiological studies have yet to be complemented by computational analyses of its molecular evolution and structural models of all its functional domains to explain the molecular basis for its conservation profile, its site-specific interactions and the propensity to conformational disorder and aggregate formation. RESULTS: We systematically annotated public sequence data to reconstruct unspliced MAPT, MAP2 and MAP4 transcripts spanning all represented genomes. Bayesian and maximum likelihood phylogenetic analyses, genetic linkage maps and domain architectures distinguished a nonvertebrate outgroup from the emergence of MAP4 and its subsequent ancestral duplication to MAP2 and MAPT. These events were coupled to other linked genes such as KANSL1L and KANSL and may thus be consequent to large-scale chromosomal duplications originating in the extant vertebrate genomes of hagfish and lamprey. Profile hidden Markov models (pHMMs), clustered subalignments and 3D structural predictions defined potential interaction motifs and specificity determining sites to reveal distinct signatures between the four homologous microtubule binding domains and independent divergence of the amino terminus. CONCLUSION: These analyses clarified ambiguities of MAPT nomenclature, defined the order, timing and pattern of its molecular evolution and identified key residues and motifs relevant to its protein interaction properties and pathogenic role. Additional unexpected findings included the expansion of cysteine-containing, microtubule binding domains of MAPT in cold adapted Antarctic icefish and the emergence of a novel multiexonic saitohin (STH) gene from repetitive elements in MAPT intron 11 of certain primate genomes.

RNA protein granules modulate tau isoform expression and induce neuronal sprouting.

The neuronal microtubule-associated protein Tau is expressed in different variants, and changes in Tau isoform composition occur during development and disease. Here, we investigate a potential role of the multivalent tau mRNA-binding proteins G3BP1 and IMP1 in regulating neuronal tau expression. We demonstrate that G3BP1 and IMP1 expression induces the formation of structures, which qualify as neuronal ribonucleoprotein (RNP) granules and concentrate multivalent proteins and mRNA. We show that RNP granule formation leads to a >30-fold increase in the ratio of high molecular weight to low molecular weight tau mRNA and an ∼12-fold increase in high molecular weight to low molecular weight Tau protein. We report that RNP granule formation is associated with increased neurite formation and enhanced process growth. G3BP1 deletion constructs that do not induce granule formation are also deficient in inducing neuronal sprouting or changing the expression pattern of tau. The data indicate that granule formation driven by multivalent proteins modulates tau isoform expression and suggest a morphoregulatory function of RNP granules during health and disease.

A refined reaction-diffusion model of tau-microtubule dynamics and its application in FDAP analysis.

Fluorescence decay after photoactivation (FDAP) and fluorescence recovery after photobleaching (FRAP) are well established approaches for studying the interaction of the microtubule (MT)-associated protein tau with MTs in neuronal cells. Previous interpretations of FDAP/FRAP data have revealed dwell times of tau on MTs in the range of several seconds. However, this is difficult to reconcile with a dwell time recently measured by single-molecule analysis in neuronal processes that was shorter by two orders of magnitude. Questioning the validity of previously used phenomenological interpretations of FDAP/FRAP data, we have generalized the standard two-state reaction-diffusion equations by 1), accounting for the parallel and discrete arrangement of MTs in cell processes (i.e., homogeneous versus heterogeneous distribution of tau-binding sites); and 2), explicitly considering both active (diffusion upon MTs) and passive (piggybacking upon MTs at rates of slow axonal transport) motion of bound tau. For some idealized cases, analytical solutions were derived. By comparing them with the full numerical solution and Monte Carlo simulations, the respective validity domains were mapped. Interpretation of our FDAP data (from processes of neuronally differentiated PC12 cells) in light of the heterogeneous formalism yielded independent estimates for the association (∼2 ms) and dwell (∼100 ms) times of tau to/on a single MT rather than in an MT array. The dwell time was shorter by orders of magnitude than that in a previous report where a homogeneous topology of MTs was assumed. We found that the diffusion of bound tau was negligible in vivo, in contrast to an earlier report that tau diffuses along the MT lattice in vitro. Methodologically, our results demonstrate that the heterogeneity of binding sites cannot be ignored when dealing with reaction-diffusion of cytoskeleton-associated proteins. Physiologically, the results reveal the behavior of tau in cellular processes, which is noticeably different from that in vitro.

The Rab GTPase Ypt7 is linked to retromer-mediated receptor recycling and fusion at the yeast late endosome.

Organelles of the endomembrane system need to counterbalance fission and fusion events to maintain their surface-to-volume ratio. At the late mammalian endosome, the Rab GTPase Rab7 is a major regulator of fusion, whereas the homologous yeast protein Ypt7 seems to be restricted to the vacuole surface. Here, we present evidence that Ypt7 is recruited to and acts on late endosomes, where it affects multiple trafficking reactions. We show that overexpression of Ypt7 results in expansion and massive invagination of the vacuolar membrane, which requires cycling of Ypt7 between GDP- and GTP-bound states. Invaginations are blocked by ESCRT, CORVET and retromer mutants, but not by autophagy or AP-3 mutants. We also show that Ypt7-GTP specifically binds to the retromer cargo-recognition subcomplex, which--like its cargo Vps10--is found on the vacuole upon Ypt7 overproduction. Our data suggest that Ypt7 functions at the late endosome to coordinate retromer-mediated recycling with the fusion of late endosomes with vacuoles.

Chronic Presence of Oligomeric Aß Differentially Modulates Spine Parameters in the Hippocampus and Cortex of Mice With Low APP Transgene Expression.

Alzheimer's disease is regarded as a synaptopathy with a long presymptomatic phase. Soluble, oligomeric amyloid-ß (Aß) is thought to play a causative role in this disease, which eventually leads to cognitive decline. However, most animal studies have employed mice expressing high levels of the Aß precursor protein (APP) transgene to drive pathology. Here, to understand how the principal neurons in different brain regions cope with moderate, chronically present levels of Aß, we employed transgenic mice expressing equal levels of mouse and human APP carrying a combination of three familial AD (FAD)-linked mutations (Swedish, Dutch, and London), that develop plaques only in old age. We analyzed dendritic spine parameters in hippocampal and cortical brain regions after targeted expression of EGFP to allow high-resolution imaging, followed by algorithm-based evaluation of mice of both sexes from adolescence to old age. We report that Aß species gradually accumulated throughout the life of APPSDL mice, but not the oligomeric forms, and that the amount of membrane-associated oligomers decreased at the onset of plaque formation. We observed an age-dependent loss of thin spines under most conditions as an indicator of a loss of synaptic plasticity in older mice. We further found that hippocampal pyramidal neurons respond to increased Aß levels by lowering spine density and shifting spine morphology, which reached significance in the CA1 subfield. In contrast, the spine density in cortical pyramidal neurons of APPSDL mice was unchanged. We also observed an increase in the protein levels of PSD-95 and Arc in the hippocampus and cortex, respectively. Our data demonstrated that increased concentrations of Aß have diverse effects on dendritic spines in the brain and suggest that hippocampal and cortical neurons have different adaptive and compensatory capacity during their lifetime. Our data also indicated that spine morphology differs between sexes in a region-specific manner.

DMSO modulates CNS function in a preclinical Alzheimer's disease model.

DMSO has a widespread use as a vehicle for water-insoluble therapeutic drug candidates but may also exert disease-relevant pharmacological effects by itself. However, its influence on the CNS has hardly been addressed. Here we examined the brain structure and function following chronic exposure to low DMSO dose at a paradigm with flawed synaptic connectivity in a preclinical transgenic mouse model for Alzheimer's disease (APPSDL mice). DMSO treatment increased spine density in a region-specific manner in the hippocampus of APPSDL mice ex vivo and in vivo. Moreover, DMSO exhibited clear influence on the behavior of this mouse line by enhancing hippocampal-dependent spatial memory accuracy, modulating hippocampal-independent olfactory habituation and displaying anxiolytic effect. Despite that most of the action of DMSO was observed in animals with elevated Aß levels, the drug did not exert its function via decreasing the oligomeric Aß species. However, challenging organotypic hippocampal slice cultures with NMDA receptor antagonist MK-801 recapitulated the effect of DMSO on spine density, indicating a tuning influence of DMSO on receptor signalization. Our findings demonstrate that DMSO should be considered as a true bioactive compound, which has the potential to be a beneficial adjuvant to counteract Aß-mediated synaptotoxicity and behavioral impairment.

Presence of a carboxy-terminal pseudorepeat and disease-like pseudohyperphosphorylation critically influence tau's interaction with microtubules in axon-like processes.

A current challenge of cell biology is to investigate molecular interactions in subcellular compartments of living cells to overcome the artificial character of in vitro studies. To dissect the interaction of the neuronal microtubule (MT)-associated protein tau with MTs in axon-like processes, we used a refined fluorescence decay after photoactivation approach and single-molecule tracking. We found that isoform variation had only a minor influence on the tau-MT interaction, whereas the presence of a C-terminal pseudorepeat region (PRR) greatly increased MT binding by a greater-than-sixfold reduction of the dissociation rate. Bioinformatic analysis revealed that the PRR contained a highly conserved motif of 18 amino acids. Disease-associated tau mutations in the PRR (K369I, G389R) did not influence apparent MT binding but increased its dynamicity. Simulation of disease-like tau hyperphosphorylation dramatically diminished the tau-MT interaction by a greater-than-fivefold decrease of the association rate with no major change in the dissociation rate. Apparent binding of tau to MTs was similar in axons and dendrites but more sensitive to increased phosphorylation in axons. Our data indicate that under the conditions of high MT density that prevail in the axon, tau's MT binding and localization are crucially affected by the presence of the PRR and tau hyperphosphorylation.

Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons.

The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP-tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin-microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43.

High-resolution imaging and evaluation of spines in organotypic hippocampal slice cultures.

Dendritic spines act as sites of excitatory neuronal input in many types of neurons. Spine shape correlates with the strength and maturity of synaptic contacts. Thus, evaluation of spine morphology is relevant for studies on neuronal development, for determination of morphological correlates of learning and memory, and for analysis of mechanisms of neurodegeneration. Here, we describe a method to determine spine morphology in an ex vivo model of organotypic hippocampal slice cultures prepared from transgenic or non-transgenic mice. Spines are imaged using confocal high-resolution imaging and evaluated by algorithm-based analysis. The approach permits semiautomated determination of spine density and classification of different spine types in dendritic segments from hippocampal subregions to evaluate intrahippocampal connectivity.